Cytogenetic study of heat stable enterotoxin (a) produced by enterotoxigenic Escherichia coli on colorectal cancer patients and its effect on cancer cells. +CD

This study was conducted for investigating the effect of heat stable enterotoxin a (STa) produced by enterotoxigenic Escherichia coli on the proliferation of colorectal cancer cells. A total of (25) stool samples were collected from patients suffering from diarrhea to isolate E. coli strains that produce STa, and after performing microscopic examination, cultural characterization and biochemical identification only (11) isolates showed positive E. coli. STa activity was estimated by using suckling mouse assay (SMA) and from these (11) isolates only (5) showed STa activity and the one with the highest STa activity was selected for large scale production of STa, which was followed by partial purification using ion-exchange chromatography, and after purification, the yield of toxin-protein was estimated as (1.08) mg/ml. The specific activity varied from (350) U/mg protein at the first step of purification to (2366.6) U/mg protein at the final step, while the final purification of the toxin was about (6.76) fold and with a yield of (18.25) . Partially purified STa was then undergoing three successive different experiments; and as follows: Experiment one: Evaluating the cytogenetic effects of STa treatment by using five different doses (100, 200, 400, 800 and 1600µg/Kg) in comparison with negative (phosphate buffer saline / PBS) and positive (mitomycin C/ MMC, at doses of 2 and 5µg/Kg) controls on mouse bone marrow cells (in vivo) by employing the following parameters: mitotic index, chromosomal aberrations and micronucleus, also, the serum level of liver functional enzymes (GOT, GPT, ALP) after oral administration of STa for five successive days, was determined. In addition, lethal dose 50 (LD 50) with certain clinicopathological changes in five organs (colon, kidney, liver, stomach and lung) was also determined after oral administration of STa for ten successive days and at two doses (500 and 1000 µg/Kg). Results showed that, none of the five different doses of STa caused any significant changes in the three examined cytogenetic parameters in the mouse bone marrow cells; precisely, neither the low dose nor the high one of STa caused reduction or induction in these parameters. In fact, clear effect in decreasing mitotic activity and increasing spontaneous frequencies of both chromosomal aberrations and micronucleus was revealed after MMC treatment. Furthermore, significant differences in mouse serum level of the three enzymes were not seen at any doses of STa, while significant reduction in the levels of these enzymes was noticed after treatment with the two doses of MMC. In this study the LD 50 test was used to investigate the lethal effect of the partially purified STa, and it was shown to be not lethal to mice at both doses of (500 and 1000) µg/Kg, since death was not recorded, moreover, no clinicopathological effects were indicated in the all examined mouse tissues, however the only noticed clinical sign was diarrhea, which was observed after three days of STa treatment. Experiment two: Evaluating the cytogenetic effects of STa treatment by using five different concentrations (100, 200, 400, 800 and 1600µg/ml) in comparison with negative (PBS) and positive (MMC, at concentration of 5µg/ml) controls on human blood lymphocytes (in vitro) obtained from both (10) normal healthy persons and (20) colorectal cancer patients by employing the following parameters: mitotic index, blast index, chromosomal aberrations and micronucleus. On the human blood lymphocytes obtained from normal healthy persons, results showed that STa, and within all the different used concentrations, did not cause any significant cytogenetic changes in the all studied cytogenetic parameters. While on the human blood lymphocytes obtained from patients with colorectal cancer, STa was shown to cause significant decrease in both mitotic and blast index, and especially at both concentrations of (800 and 1600µg/ml) and this decrease was concentration dependent, but at the same time non significant changes were seen in both chromosomal aberrations and micronucleus parameters and for the all used concentrations. However, reduced mitotic and blast index and induced chromosomal aberrations and micronucleus frequencies of human blood lymphocytes that were obtained from both normal healthy persons and colorectal cancer patients, were observed after treatment with MMC. Experiment three: Evaluating the cytotoxic effect of STa treatment on primary cancer cell cultures, obtained from tumor samples that were collected from (13) cancer patients and as follows: (five colon cancer patients, two bladder cancer patients, two breast cancer patients, two stomach cancer patients and two lung cancer patients), and on normal cell line (rat embryonic fibroblast / REF) (in vitro) with the use of different concentrations starting from (1) mg/ml and ending with (0.0002) mg/ml by making two fold serial dilutions by using the 96- well microtiter plate, and in comparison with negative (PBS) and positive (MMC, at concentration of 10 µg/ml) controls . Results showed that, after (24) hours of exposure to STa, the growth of all primary cancer cell cultures obtained from colon cancer patients was inhibited by STa treatment and this inhibition was concentration dependent. Also it was shown that the cytotoxic effect of the high concentration of STa was close to that seen after MMC treatment. While no differences were seen in the growth of all primary cancer cell cultures that were obtained from the other cancer patients, which mean that STa treatment neither inhibit nor enhanced their growth. At the same time STa did not show or has any cytotoxic effect on the normal cell line (REF).